1. Field of the Invention
The invention relates to novel polynucleotides coding for a polypeptide with citrate synthase activity, bacteria containing the polynucleotides and polypeptides and methods of production of amino acids using these bacteria.
2. Discussion of the Background
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
Amino acids are used in human medicine, in the pharmaceutical industry, in the food industry and quite particularly in animal nutrition.
Amino acids may be produced by fermentation of strains of coryneform bacteria, preferably Corynebacterium glutamicum. Owing to their great importance, work is constantly in progress for improving the production processes. Process improvements may relate to the fermentation technology, for example, stirring and supply of oxygen, or to the composition of the nutrient media, for example, the sugar concentration during fermentation, or processing to the product form by, for example, ion-exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
Methods of mutagenesis, selection and mutant screening are employed for improving the performance characteristics of these microorganisms. In this way we obtain strains that are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance, and which produce amino acids. A known antimetabolite is the lysine analog S-(2-aminoethyl)-L-cysteine (AEC).
Methods of recombinant DNA technology have also been used for some years now for strain improvement of L-amino acid-producing strains of the genus Corynebacterium, preferably Corynebacterium glutamicum, by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production.
Synoptic descriptions of the biology, genetics and biotechnology of Corynebacterium glutamicum are given in “Handbook of Corynebacterium glutamicum” (Eds.: L. Eggeling and M. Bott, CRC Press, Taylor & Francis, 2005), in the special issue of the Journal of Biotechnology (Chief Editor: A. Pühler) with the title “A new era in Corynebacterium glutamicum biotechnology” (Journal of Biotechnology 104/1-3, (2003)) and in the book by T. Scheper (Managing Editor) “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering/Biotechnology 79, Springer Verlag, Berlin, Germany, 2003).
The nucleotide sequence of the genome of Corynebacterium glutamicum is described in Ikeda and Nakagawa (Applied Microbiology and Biotechnology 62, 99-109 (2003)), in EP 1 108 790 and in Kalinowski et al. (Journal of Biotechnology 104/1-3, 2003)).
The nucleotide sequence of the genome of Corynebacterium efficiens is described in Nishio et al. (Genome Research, 13 (7), 1572-1579 (2003)).
The nucleotide sequences of the genome of Corynebacterium glutamicum and Corynebacterium efficiens are also available in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA), in the DNA Data Bank of Japan (DDBJ, Mishima, Japan) or in the nucleotide sequence database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).
The wild-type sequence of the coding region of the gltA gene of Corynebacterium glutamicum is presented in SEQ ID NO: 1 in the specification of the present application. In addition, the sequences located upstream and downstream of the coding region are shown in SEQ ID NO: 3 and 25. The amino acid sequence of the encoded GltA polypeptide (citrate synthase) is accordingly given in SEQ ID NOs: 2, 4 and 26.